New nitric oxide or hydrogen sulfide releasing aspirins

Article abstract of DOI:10.1021/jm2004514

A new series of (((R-oxy)carbonyl)oxy)methyl esters of aspirin (ASA), bearing nitric oxide (NO) or hydrogen sulfide (H₂S) releasing groups, was synthesized, and the compounds were evaluated as new ASA co-drugs. All the products were quite stabl

Full text of DOI:10.1021/jm2004514

ARTICLE
pubs.acs.org/jmc
New Nitric Oxide or Hydrogen Sulfide Releasing Aspirins
Loretta Lazzarato, Konstantin Chegaev, Elisabetta Marini, Barbara Rolando, Emily Borretto,
Stefano Guglielmo, Sony Joseph, Antonella Di Stilo, Roberta Fruttero, and Alberto Gasco*
Dipartimento di Scienza e Tecnologia del Farmaco, Universitꢀa degli Studi di Torino, Via Pietro Giuria 9, 10125 Torino, Italy
S Supporting Information
b
ABSTRACT: A new series of (((R-oxy)carbonyl)oxy)methyl esters of aspirin (ASA),
bearing nitric oxide (NO) or hydrogen sulfide (H2S) releasing groups, was synthesized, and
the compounds were evaluated as new ASA co-drugs. All the products were quite stable in
buffered solution at pH 1 and 7.4. Conversely, they were all rapidly metabolized, producing
ASA and the NO/H2S releasing moiety used for their preparation. Consequent on ASA
release, the compounds were capable of inhibiting collagen-induced platelet aggregation of
human platelet-rich plasma (PRP). The simple NO/H2S donor substructures were able to relax contracted rat aorta strips, with a
NO- and H2S-dependent mechanism, respectively, but they either did not trigger antiaggregatory activity or displayed antiplatelet
potency markedly below that of the related co-drug. The new products might provide a safer and improved alternative to the use of
ASA principally in its anti-inflammatory and antithrombotic applications.
’ INTRODUCTION
enzymatic lability of the acetyloxy group.17 A number of similar
products have been developed, but to our knowledge, few
well-documented examples of “true” ASA NO-donor co-drugs
are known,18,19 and no true ASA H2S-donor co-drug has yet
been described. This paper reports the synthesis of a new class
of aryloxy and alkyloxy carbonyloxymethyl esters of ASA, bear-
ing either nitrooxy NO-donor moieties (9aÀd, Scheme 1) or
the H2S-donor residue (3-thioxo-3H-1,2-dithiol-5-yl) (16a,b,
Scheme 2). All these products are shown to be stable in acid
and physiological pH solutions but are able to release aspirin
when incubated in human serum. The antiaggregatory prop-
erties of the products, and the vasodilator effects of the simple
NO-/H2S-donor moieties used for their preparation, are also
discussed.
Although it was introduced onto the market over 100 years
ago, the adverse gastrointestinal effects of aspirin (ASA, acet-
ylsalicylic acid (1), Chart 1), which is the most widely used
nonsteroidal anti-inflammatory drug (NSAID), are still a sig-
nificant drawback to its use.1À3 A number of strategies have been
proposed to overcome this problem, including the “gaseous
solution”.2,4,5 This approach consists of conjugating a nitric oxide
(NO) or a hydrogen sulfide (H2S) releasing moiety with ASA via
an ester link. NO is an important gaseous messenger that
mediates a variety of physiological actions, including gastro-
protection.6 NO defends the gastric mucosa against adverse
effects consequent on NSAID-induced COX-1 inhibition, in-
cluding decreased mucosal blood flow, reduced mucus and
bicarbonate secretion, promotion of neutrophil adherence and
activation, and modulation of inflammatory mediators.7À9 In
addition it is a key determinant of vascular health exerting
antiplatelet, antithrombotic, and vasodilating effects.10 In recent
years, H2S has also been recognized as an important gaseous
signaling molecule. Like NO, it possesses a “dual personality”,
since at endogenous concentrations it displays a variety of
beneficial effects but is detrimental at superphysiological con-
centrations. It shares many biological activities with NO, includ-
ing reduction of neutrophil adhesion, modulation of inflam-
matory mediator release, protection against gastric injury, and
beneficial effects on cardiovascular system including vasodilator
activity.4,11À14 The NicOx NO-donor aspirins 2, 3, 4, 5 and the
H2S-donor CTG Pharma aspirin 6 (Chart 1), which are the
prototypes of this class of drugs, were designed following the
aforementioned approach.15,16 The drawback of these products
is that they are rapidly metabolized in human plasma into
salicylates and then into salicylic acid and NO/H2S releasing
moieties, without any formation of ASA. This is due to the loss
of negative charge on the ASA moiety, which induces high
’ RESULTS AND DISCUSSION
Chemistry. The synthetic routes used to prepare the ASA NO-
donor co-drugs are summarized in Scheme 1. Chloromethyl
chloroformate was treated, in CH2Cl2 solution, in the presence of
pyridine (Py), with the appropriate NO-donor alcohols 7a,b and
phenols 7c,d. The resulting chloromethylcarbonates 8aÀd were
used for the next reaction with ASA, in the presence of Cs2CO3
in DMF, to give the final carbonates 9aÀd. The preparation of
ASA H2S-donor co-drugs 16a,b (Scheme 2) required the use of
the known H2S-donor 5-(4-hydroxyphenyl)-3H-1,2-dithiole-3-
thione (10)14 and of the new intermediates 12 and 14. The
former product was synthesized treating 10 with O-tetrahydro-
pyranyl (THP) protected ethylene glycol under Mitsunobu
conditions (triphenylphosphine (PPh3), diisopropyl azodicar-
boxylate (DIAD)). The THP group was removed under the
Received: April 26, 2011
Published: June 21, 2011
r
2011 American Chemical Society
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Chart 1. Examples of NO-Donors and H2S-Donor Aspirin
Scheme 1a
a Conditions: (i) Py, CH2Cl2, À15 °C; (ii) ASA, Cs2CO3, DMF, room temp.
Scheme 2a
Scheme 3. Possible Hydrolytic Routes of Compounds
(13) in the presence of Cs2CO3 in DMF. The resulting product
14 was treated with neat SO2Cl2 to give 15. Finally, reaction of 15
with phenol 10 or alcohol 12 in the presence of 4-methylmor-
pholine in CH2Cl2 gave the desired carbonates 16a and 16b.
Hydrolysis Studies. The possible hydrolytic routes of the new
carbonates are reported in Scheme 3. In order to be true ASA
NO/H2S donor co-drugs, the products must have a rate constant
of deacetylation k2 slower than the hydrolytic constant k1. The
stability of all the compounds was assessed by high-performance
liquid chromatography (HPLC) in buffered solution at pH 1.0
and 7.4, as well as in human serum. The results are reported in
Table 1. All the products remained more than 96% unchanged
after 3 h of incubation in acid solution. The same occurred at
physiological pH, with the exception of 9b and 16a, which were
transformed more extensively (85% and 80% unchanged,
respectively). This was not the case in human serum, in
which it is known that a variety of esters are hydrolyzed by
a Conditions: (i) PPh3, DIAD, THF dry, À15 °C to room temp; (ii)
PPTS, MeOH, 55 °C; (iii) Cs2CO3, DMF, room temp; (iv) SO2Cl2; (v)
10 or 12, 4-methylmorpholine, CH2Cl2, À15 °C.
action of pyridinium p-toluensulfonate (PPTS) in methanol to
give the expected 12. The latter intermediate was obtained by
reaction of ASA with O-(chloromethyl)-S-ethyl thiocarbonate
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Table 1. Stability of the Compounds 9aÀd, 16a, 16b in Buffered Solutions (Percentage of Unchanged Compound after 3 h) and in
Human Serum (Half-Life, Percent of Maximal Amounts of Aspirin Released, and AUC Values of Aspirin Released over the First
10 min of Incubation Time) and Antiaggregatory Activities of Compounds 9aÀd, 16a, 16b
stability in buffered solutions,
% of unchanged at 3 ha
stability in human serum
% max of
ASA releasedc
ASA AUC0À10min
platelet aggregation
compd
pH 1.0
pH 7.4
t
1/2 (min)b
(μM min)
IC50 (95% CL) (μM)
ASA
9a
90
98
98
98
98
96
98
90
98
85
98
95
80
96
63
<1
<1
1.6
2.2
2.1
3.0
54 (49À60)
9
25
40
34
36
42
84
227
286
260
247
260
194 (182À208)
93 (82À104)
68 (60À77)
9b
9c
9d
103 (93À114)
150 (134À168)
16 (13-21)
16a
16b
a SEM e 1%. b SEM e 0.1. c SEM e 1.5%.
carboxylesterases:20 both the nitrogen and the sulfur containing
compounds were very rapidly hydrolyzed, following pseudo-first-
order kinetics. The observed pseudo-first-order rate constants
(kobs) and the half-lives (t1/2, Table 1) were determined by fitting
the data to one-phase exponential decay equation (GraphPad
Prism software, version 5).
As shown in Scheme 3, ASA, R-oxycarbonyloxymethyl esters
of salicylic acid (salicylates), related hydroxy derivatives, and
salicylic acid were detected during hydrolysis in human serum.
For all the compounds, the final metabolites were salicylic acid
and hydroxyl derivatives. The time-course of the degradation
products, monitored over 10 min and 4 h of incubation time, in
the case of compound 16a, is reported in Figure 1 as an example.
The peak amounts of ASA detected for each compound, ex-
pressed as % of the initial carbonate concentration, are given in
Table 1, together with the areas under the ASA concentrationÀ
time curves, measured for each compound over the first 10 min of
incubation (AUC0À10). Among the nitrooxy-containing com-
pounds, the aromatic carbonates 9c and 9d released ASA more
extensively than the aliphatic compounds (9a and 9b). Both the
sulfur-containing esters 16a and 16b were quite good ASA
producers. In conclusion, all the products act as true ASA co-
drugs.
Platelet Antiaggregatory Activity. Antiaggregatory effects
of the new ASA co-drugs were studied on collagen induced
platelet aggregation of human rich plasma (PRP), taking aspirin
as reference standard. The inhibitory activity was assessed by
adding each product to PRP 10 min before addition of the
stimulus. The calculated antiaggregatory potencies (IC50) are
reported in Table 1. The NO-donor structures 7a,b,d, and the
H2S-donor structure 10, used to hybridize ASA, did not trigger
any antiaggregatory action when tested at 300 μM, under the
same conditions used to test the corresponding co-drugs.
Alcohols 7c and 12 acted slightly differently, showing 163 and
184 μM IC50 values, respectively, potencies markedly below
those of the related co-drugs 9c and 16b. Consequently, the
antiaggregatory activities of all the products may reasonably be
principally attributed to their capacity to release ASA.
Figure 1. Time-course of the degradation products of compound 16a at
4 h (a) and at 10 min (b) incubation time in human serum. Values are the
mean ( SEM (SEM e 3; number of determinations is 3).
The antiaggregatory IC50 values versus AUC values for the
carbonates object of the present research fit this line quite well;
the only exception is 16b (Figure 2). Synergism between ASA
and alcohol 12 could be responsible for this difference: when the
antiaggregatory potency was evaluated using 1:1 mixtures of the
products, at a concentration at which both compounds separately
are inactive (10 μM, mol/mol), a significant inhibition of platelet
aggregation (∼66%) was observed.
Vasodilator Activities. The vasodilator activity of the NO-
donor moieties 7aÀd, used to hybridize ASA, was evaluated on
endothelium denuded rat aorta strips precontracted with phe-
nylephrine. All the products caused relaxation of the contracted
tissue in a concentration-dependent manner. Their potencies,
expressed as EC50, are in Table 2. As expected, both among the
In a previous study, we showed that the areas under the ASA
release-time curves (AUC0À10) in human serum, measured for a
series of (nitrooxyacyloxy)methyl esters of ASA, correlate
linearly with the corresponding antiaggregatory potencies.18
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ARTICLE
related co-drug. By contrast, they display NO-dependent and
H2S-dependent vasodilator activities, respectively. In view of the
role exerted by NO and H2S in the gastrointestinal tract and in
the cardiovascular system, as well as in the regulation of the
inflammatory processes, this new class of ASA co-drugs might
represent a safer and improved alternative to ASA in the
antithrombotic and anti-inflammatory therapies.
’ EXPERIMENTAL SECTION
1
Chemistry. H and 13C NMR spectra were recorded on a Bruker
Avance 300 at 300 and 75 MHz, respectively, using SiMe4 as internal
standard. Low resolution mass spectra were recorded with a Finnigan-
Mat TSQ-700. Melting points were determined with a capillary appa-
ratus (B€uchi 540). Flash column chromatography was performed on
silica gel (Merck Kieselgel 60, 230À400 mesh ASTM); PE stands for
40À60 petroleum ether. The progress of the reactions was followed by
thin layer chromatography (TLC) on 5 cm  20 cm plates with a layer
thickness of 0.2 mm. Anhydrous magnesium sulfate was used as drying
agent for the organic phases. Organic solvents were removed under
vacuum at 30 °C. Elemental analyses (C, H, N) of the target compounds
were performed by Section de Pharmacie, Service de Microanalyse
(Geneva), and the results are within 0.4% of the theoretical values.
Target compounds were prepared, as assessed using the aforementioned
standard spectroscopic techniques and elemental analyses, in g95%
purity. Compounds 7a,21 7b,22 7c,23 7d,24 10,25 and 1326 were obtained
as described elsewhere.
General Procedure for the Preparation of 8aÀd. To a
solution of the appropriate alcohol or phenol (2.5 mmol) and chloro-
methyl chloroformate (0.25 mL, 2.7 mmol) in dry CH2Cl2 (15 mL),
stirred at À15 °C, a solution of Py (0.22 mL, 2.7 mmol) in dry CH2Cl2
(10 mL) was added dropwise. At the end of the addition, the iceÀsalt
bath was removed and the reaction mixture allowed to reach room
temperature. After 15 min, the solvent was removed and the resulting oil
was purified by flash chromatography. Chromatographic eluents and
yields of the products were as follows.
Figure 2. Antiaggregatory IC50 values versus AUC0À10 values of aspirin
released in human serum from compounds 9aÀd, 16a, 16b on the linear
correlation between antiaggregatory IC50 values and AUC0À10 values of
aspirin released in human serum obtained in a previous study (ref 16)
from a series of (nitrooxyacyloxy)methyl esters of ASA.
Table 2. Vasodilator Activities of Compounds 7aÀd, 10,
and 12
vasodilator activity (EC50 ( SEM) (μM)
compd
with inhibitor
7a
7b
7c
7d
10
12
4.0 ( 0.6a
1.7 ( 0.3a
1.0 ( 0.2a
0.13 ( 0.03a
5.9 ( 0.6c
8.0 ( 0.5c
>100 μMa,b
>100 μMa,b
>100 μMa,b
>100 μMa,b
17 ( 1c,d
16 ( 2c,d
a Aorta strips precontracted with 1 μM phenylephrine. b Inhibitor:
1 μM ODQ. c Aorta strips precontracted with 25 mM KCl. d Inhibitor:
10 μM glibenclamide.
aliphatic and among the aromatic carbonates, the dinitrooxy-
substituted products are more potent than their mononitrooxy
analogues. The vasodilator effect was abolished by the presence
of 1 μM ODQ (1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one),
a well-known inhibitor of the soluble guanylate cyclase (sGC), in
keeping with NO-induced activation of this enzyme’s being the
mechanism underlying the effect. The vasodilator actions of the
H2S releasing products 10 and 12 were evaluated on endothe-
lium denuded rat aorta strips precontracted with KCl. The two
alcohols showed similar vasodilator potencies but lower than
those of the related nitrooxy carbonates (Table 2). In experi-
ments performed in the presence of glibenclamide, a well-known
potent blocker of ATP-modulated K+-channels, the doseÀ
response curves obtained for the two compounds were signifi-
cantly shifted rightward. This is consistent with the involvement
of H2S in the vasodilator action of the compounds.
Chloromethyl-3-nitrooxypropyl Carbonate (8a). Eluent
1
(PE/CH2Cl2 7/3 v/v); colorless oil; yield 83%. H NMR (CDCl3)
δ 2.15 (qi, 2H, ÀCH2CH2ONO2), 4.35 (t, 2H, ÀOCH2CH2À), 4.58
(t, 2H, ÀCH2ONO2), 5.74 (s, 2H, ÀCH2Cl). 13C NMR (CDCl3) δ
26.3, 64.9, 69.1, 72.3, 153.2.
Chloromethyl-2,3-bis(nitrooxy)propyl Carbonate (8b). Elu-
ent (PE/CH2Cl2 1/1 v/v); colorless oil; yield 80%. 1H NMR (CDCl3)
δ 4.44 (dd, 1H, ÀCHHOÀ), 4.56À4.70 (m, 2H, ÀCHHONO2 +
ÀCHHOÀ), 4.81 (dd, 1H, ÀCHHONO2), 5.48À5.54 (m, 1H,
ÀCHONO2), 5.72À5.77 (m, 2H, ÀCH2Cl). 13C NMR (CDCl3) δ
64.6, 68.2, 72.6, 75.6, 152.9.
Chloromethyl-4-(3-nitrooxypropyl)phenyl Carbonate (8c).
Eluent (PE/CH2Cl2 8/2 v/v); colorless oil, which solidified on stand-
ing in the freezer; yield 75%. 1H NMR (CDCl3) δ 2.05 (m, 2H,
ÀCH2CH2ONO2), 2.75 (t, 2H, ÀCH2CH2CH2ONO2), 4.45 (t, 2H,
ÀCH2ONO2), 5.82 (s, 2H, ÀCH2Cl), 7.13À7.26 (m, 4H, C6H4). 13
C
NMR (CDCl3) δ 28.3, 31.1, 72.1, 72.5, 120.9, 129.5, 138.6, 149.2, 152.1.
4-(2,3-Bis(nitrooxy)propyl)phenylchloromethyl Carbonate
(8d). Eluent (PE/CH2Cl2 6/4 v/v); yellowish oil; yield 50%. 1H
NMR (CDCl3) δ 2.99À3.14 (m, 2H, ÀCH2CHÀ), 4.44 (dd, 1H,
ÀCHHONO2), 4.73 (dd, 1H, ÀCHHONO2), 5.40À5.47 (m, 1H,
’ CONCLUSIONS
We were able to develop a new class of NO-donor and, for the
first time, of true H2S-donor ASA co-drugs. All the products are
capable of fast ASA release when incubated in human serum,
following pseudo-first-order kinetics. By contrast, they are rather
stable in acid and physiological pH. They inhibit collagen-
induced platelet aggregation of human platelet-rich plasma.
The simple NO-donor and H2S-donor moieties, used to prepare
the final products, do not trigger antiaggregatory activity or
display antiplatelet potencies definitively lower than that of the
ÀCHONO2), 5.82 (s, 2H, ÀCH2Cl), 7.20À7.30 (m, 4H, C6H4). 13
C
NMR (CDCl3) δ 34.9, 70.0, 72.6, 79.2, 121.5, 130.5, 132.6, 150.2, 151.9.
5-(4-(2-Hydroxyethoxy)phenyl)-3H-1,2-dithiole-3-thione
(12). To a solution of Ph3P (0.28 g, 1.1 mmol) in dry THF (10 mL),
stirred under positive nitrogen pressure at À15 °C, DIAD (0.22 mL, 1.1
mmol) was added. The reaction mixture was stirred for 15 min until a
white precipitate formed, and 10 (0.20 g, 0.90 mmol) was added,
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followed by 2-(tetrahydropyran-2-yloxy)ethanol (0.13 g, 0.90 mmol).
The resulting mixture was stirred for 24 h at room temperature, then
poured into H2O (10 mL) and extracted with Et2O (3 Â 10 mL). The
combined organic layers were washed with brine, dried, filtered, and
concentrated under reduced pressure. The crude product was puri-
fied by flash chromatography (PE/acetone 9/1 v/v) to give 5-(4-
(2-(tetrahydropyran-2-yloxy)ethoxy)phenyl)-3H-1,2-dithiole-3-thione
(11) as a reddish-brown oil; yield 70%.1H NMR (CDCl3) δ 1.52À1.89
(m, 6H, 3CH2 pyran), 3.51À3.58 (m, 1H), 3.81À4.25 (m, 5H),
(CH2Opyran, ÀOCH2CH2OÀ), 4.70À7.72 (m, 1H, ÀOCHOÀ),
7.02 (d, 2H, C6H4), 7.40 (s, 1H, C3S3H), 7.62 (d, 2H, C6H4). 13C
NMR (CDCl3) δ 19.4, 25.4, 30.5, 62.3, 65.6, 67.8, 99.1, 115.5, 124.2,
128.6, 134.6, 162.3, 173.1, 215.1. MS (CI) m/z 355 (M + 1)+.
11 (0.44 g, 1.20 mmol) was dissolved in MeOH (15 mL), and a
catalytic amount of PPTS was added. The resulting mixture was heated
at 55 °C for 2 h, then concentrated under reduced pressure. The crude
product was purified by flash chromatography (PE/acetone 6/4 v/v) to
give a reddish solid, which was recrystallized from EtOH to give the title
compound as a yellowish-orange solid. Yield 40%; mp 117.5 °C (from
EtOH). 1H NMR (CDCl3) δ 2.12 (sbr, 1H, OH), 4.02 (t, 2H), 4.16 (t,
2H)(ÀOCH2CH2OH), 7.00 (d, 2H, C6H4), 7.39 (s, 1H, C3S3H), 7.61
(d, 2H, C6H4). 13C NMR (CDCl3) δ 61.2, 69.6, 115.5, 124.5, 128.6,
134.7, 162.0, 172.9, 215.1. MS (CI) m/z 271 (M + 1)+.
General Procedure for the Preparation of 9aÀd and 14. To
a solution of acetylsalicylic acid (0.22 g, 1.2 mmol) in DMF (5 mL) was
added Cs2CO3 (0.20 g, 0.60 mmol), and the resulting mixture was
vigorously stirred for 15 min. The appropriate amount of chloromethyl
carbonate (1.0 mmol) was then added, and the reaction mixture was
stirred at room temperature for 24 h. The reaction mixture was diluted
with Et2O (25 mL) and washed with H2O, saturated solution of
NaHCO3, and brine. The organic layer was dried, filtered, and con-
centrated under reduced pressure. The crude product thus obtained was
purified by flash chromatography. Chromatographic eluents and yields
of the products are listed below.
((3-Nitrooxypropyl)carbonyl)oxymethyl 2-(Acetyloxy)-
benzoate (9a). Eluent (PE/EtOAc 9/1 v/v); colorless oil; yield 75%.
1H NMR (CDCl3) δ: 2.12 (qi, 2H, ÀCH2CH2ONO2), 2.36 (s, 3H,
ÀCH3), 4.31 (t, 2H, ÀOCH2CH2À), 4.55 (t, 2H, ÀCH2ONO2), 5.95
(s, 2H, ÀOCH2OÀ), 7.12 (d, 1H), 7.34 (t, 1H), 7.61 (t, 1H), 8.08 (d,
1H) (C6H4). 13C NMR (CDCl3) δ: 21.0, 26.3, 64.4, 69.2, 82.2, 121.7,
124.1, 126.2, 132.5, 134.9, 151.2, 153.8, 162.7, 168.2. MS (CI) m/z 358
(M + 1)+. Anal. (C14H15NO10) C, H, N.
((2,3-Bis(nitrooxy)propyl)carbonyl)oxymethyl 2-(Acetyloxy)-
benzoate (9b). Eluent (PE/EtOAc 8/2 v/v); colorless oil; yield 29%.
1H NMR (CDCl3) δ: 2.36 (s, 3H, ÀCH3), 4.39 (dd, 1H, ÀCHHONO2),
4.54 (dd, 1H, ÀCHHONO2), 4.64 (dd, 1H, ÀCHHOÀ), 4.80 (dd,
1H, ÀCHHOÀ), 5.45À5.51 (m, 1H, ÀCHONO2), 5.97 (s, 2H,
ÀOCH2O), 7.13 (d, 1H), 7.35 (t, 1H), 7.62 (t, 1H), 8.08 (d, 1H)
(C6H4). 13C NMR (CDCl3) δ: 20.9, 64.3, 68.2, 75.7, 82.5, 121.6, 124.1,
126.3, 132.3, 135.0, 151.2, 153.5, 162.6, 169.7. MS (CI) m/z 419 (M +
1)+. Anal. (C14H14N2O13) C, H, N.
ÀCHHONO2), 5.38À5.46 (m, 1H, ÀCHONO2), 6.05 (s, 2H,
ÀOCH2OÀ), 7.13À7.38 (m, 6H), 7.61 (t, 1H), 8.11 (d, 1H)
(2C6H4). 13C NMR (CDCl3) δ 21.0, 34.9, 70.0, 79.2, 82.5, 121.7,
124.1, 126.2, 130.5, 132.3, 135.0, 150.3, 151.3, 152.5, 162.7, 169.7. MS
(CI) m/z 495 (M + 1)+. Anal. (C20H18N2O13) C, H, N.
(Ethylthiocarbonyl)oxymethyl 2-(Acetyloxy)benzoate (14).
Eluent (PE/EtOAc 8/2 v/v); colorless oil; yield 87%. 1H NMR
(CDCl3) δ: 1.33 (t, 3H, ÀCH2CH3), 2.36 (s, 3H, ÀCH3), 2.90 (q,
2H, ÀCH2CH3), 6.00 (s, 2H, ÀOCH2OÀ), 7.13 (d, 1H), 7.35 (t, 1H),
7.60 (t, 1H), 8.07 (d, 1H) (C6H4). 13C NMR (CDCl3) δ: 14.8, 21.0,
25.5, 80.5, 121.9, 124.1, 126.1, 132.3, 134.8, 151.2, 162.7, 169.6, 170.8.
MS (EI) m/z 298 (M)+.
General Procedure for the Preparation of 16a and 16b.
SO2Cl2 (0.88 mL, 10.9 mmol) was added dropwise to 14 (3.24 g, 10.9
mmol), and the mixture was stirred at 0 °C. The mixture was allowed to
reach room temperature and stirred for 1 h. The reaction mixture was
then concentrated under reduced pressure to give 15 as a colorless oil
that was used in the next synthetic step without further purification.
To a solution of 15 (0.85 g, 3.1 mmol) in CH2Cl2 (30 mL), stirred at
À15 °C, a solution of the appropriate hydroxy derivative (2.6 mmol) and
N-methylmorpholine (0.31 mL, 2.6 mmol) in CH2Cl2 (30 mL) was
added dropwise. The reaction mixture was stirred for 1 h at À15 °C, then
for 24 h at room temperature, then poured into H2O (20 mL) and
extracted with CH2Cl2 (3 Â 20 mL). The combined organic layers were
washed with brine, dried, filtered, and concentrated under reduced
pressure. The crude product was purified by flash chromatography.
Chromatographic eluents and yields of the products were as follows.
((4-(3-Thioxo-3H-1,2-dithiol-5-yl)phenoxy)carbonyloxy)-
methyl 2-(Acetyloxy)benzoate (16a). Eluent (PE/EtOAc 8/2
v/v); reddish-brown solid, which was recrystallized from EtOH to give
the title compound as a yellowish-orange solid; yield 30%; mp
1
103.5À104 °C (EtOH). H NMR (CDCl3) δ 2.37 (s, 3H, ÀCH3),
6.07 (s, 2H, ÀOCH2OÀ), 7.15 (d, 1H, C6H4), 7.33 À 7.40 (m, 4H,
2C6H4 + C3HS3), 7.61À7.71 (m, 3H), 8.13 (d, 1H) (2C6H4). 13C NMR
(CDCl3) δ 21.0, 82.6, 121.5, 122.2, 124.1, 126.2, 128.4, 129.8, 132.3,
135.0, 136.2, 151.3, 151.8, 153.3, 162.6, 169.8, 171.4, 215.5. MS (CI)
m/z 463 (M + 1)+. Anal. (C20H14O7S3) C, H, N.
((2-(4-(3-Thioxo-3H-1,2-dithiol-5-yl)phenoxy)ethoxy)car-
bonyloxy)methyl 2-(Acetyloxy)benzoate (16b). Eluent (PE/
EtOAc 75/25 v/v); reddish-brown solid, which was recrystallized from
EtOH to give the title compound as a yellowish-orange solid; yield 20%;
1
mp 105 °C (EtOH). H NMR (CDCl3) δ 2.36 (s, 3H, ÀCH3), 4.28
(t, 2H), 4.58 (t, 2H) (ÀOCH2CH2OÀ), 5.98 (s, 2H, ÀOCH2OÀ),
6.96 (d, 2H), 7.13 (d, 1H), 7.33 (t, 1H) (2C6H4), 7.37 (s, 1H, C3HS3),
7.56À7.65 (m, 3H), 8.8 (d, 1H) (2C6H4). 13C NMR (CDCl3) δ 21.0,
65.7, 66.4, 82.3, 115.6, 121.7, 124.1, 124.7, 126.2, 128.6, 132.2, 134.8,
134.9, 151.2, 153.9, 161.5, 162.6, 169.7, 172.7, 215.2. MS (CI) m/z 507
(M + 1)+. Anal. (C22H18O8S3) C, H, N.
Evaluation of Stability in Buffered Solutions and in Human
Serum. Hydrolysis in Acidic Medium (pH 1.0) and in Phosphate
Buffer (pH 7.4). A 2 mL aliquot of 0.5 mM solution of each compound in
DMSO was diluted to 10 mL using 0.1 M HCl to obtain pH 1.0 or
50 mM phosphate buffer to obtain pH 7.4. The resulting solution was
maintained at 37 ( 0.5 °C, and at appropriate time intervals, an amount
of 20 μL of the solution was analyzed by RP-HPLC. All experiments
were performed in triplicate.
Hydrolysis in Human Serum. A solution of each compound (10 mM)
in DMSO was added to human serum (sterile-filtered from human male
AB plasma, Sigma-Aldrich) preheated at 37 °C. The final concentration
of the compound was 200 μM. The resulting solution was incubated at
37 ( 0.5 °C, and at appropriate time intervals, an amount of 300 μL of
the reaction mixture was withdrawn and added to 300 μL of acetonitrile
containing 0.1% trifluoroacetic acid in order to deproteinize the serum.
The sample was sonicated, vortexed, and then centrifuged for 10 min at
(4-(3-Nitrooxypropyl)phenoxycarbonyl)oxymethyl 2-
(Acetyloxy)benzoate (9c). Eluent (PE/EtOAc 8/2 v/v); white
solid; mp 52.5À53 °C (i-Pr2O); yield 64%. 1H NMR (CDCl3) δ:
2.04 (qi, 2H, ÀCH2CH2ONO2), 2.37 (s, 3H, ÀCH3), 2.74 (t, 2H,
ÀCH2CH2CH2ONO2), 4.45 (t, 2H, ÀCH2ONO2), 6.05 (s, 2H,
ÀOCH2OÀ), 7.13À7.22 (m, 5H), 7.37 (t, 1H), 7.65 (t, 1H), 8.11 (d,
1H) (2C6H4). 13C NMR (CDCl3) δ: 21.0, 28.3, 31.1, 72.1, 82.5, 121.0,
121.7, 124.1, 126.2, 129.5, 132.3, 135.0, 138.4, 149.3, 151.2, 152.7, 162.7,
169.7. MS (CI) m/z 434 (M + 1)+. Anal. (C20H19NO10) C, H, N.
(4-(2,3-Bis(nitrooxy)propyl)phenoxycarbonyl)oxymethyl
2-(Acetyloxy)benzoate (9d). Eluent (PE/EtOAc 8/2 v/v); color-
less oil; yield 51%.1H NMR (CDCl3) δ 2.36 (s, 3H, ÀCH3), 2.97À3.13
(m, 2H, ÀCH2CHÀ), 4.43 (dd, 1H, ÀCHHONO2), 4.73 (dd, 1H,
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ARTICLE
2150g. The clear supernatant was filtered by 0.45 μm PTFE filters
(Alltech) and analyzed by RP-HPLC. All experiments were performed at
least in triplicate.
recorded by an isometric transducer connected to the MacLab System
PowerLab. Addition of the drug vehicle, DMSO, had no appreciable
effect on contraction degree. At least five experiments for each com-
pound were performed.
The reverse-phase HPLC procedure afforded separation and quanti-
tation of the remaining compounds and of the products of hydrolysis
(aspirin, salicylic acid, salicylate, and hydroxy derivatives bearing NO-
donor or H2S-donor moieties). HPLC analyses were performed with an
HP 1100 chromatographic system (Agilent Technologies, Palo Alto,
CA, U.S.) equipped with a quaternary pump (model G1311A), a
membrane degasser (G1379A), and a diode-array detector (DAD)
(model G1315B) integrated into the HP1100 system. Data analysis
was done using an HP ChemStation system (Agilent Technologies).
The injection volume was 20 μL (Rheodyne, Cotati, CA). The analytical
column was a Nucleosil 100-5C18 Nautilus (250 mm  4.6 mm, 5 μm
particle size) (Macherey-Nagel) column, eluting with a flow rate of
1.2 mL/min. The samples were analyzed using a gradient method
employing a mobile phase consisting of acetonitrile/water with 0.1%
trifluoroacetic acid 55/45 over the first 4 min, grading to 70/30 at 6 min,
maintaining 70/30 until 15 min, then returning to 55/45 at 20 min. The
column effluent was monitored at 226 nm (for compounds, aspirin and
NO-donor hydroxy derivatives), at 360 nm (for H2S-donor hydroxy
derivatives), and at 240 nm (for salicylic acid and salicylates) referenced
against a 600 nm wavelength. Quantitation was done using calibration
curves of compounds and relative metabolites chromatographed under
the same conditions. The linearity of the calibration curves was
determined in a concentration range of 1À200 μM (r2 > 0.99).
Inhibition of Platelet Aggregation in Vitro. Venous blood
samples were obtained from healthy volunteers who had not taken any
drug for at least 2 weeks. Volunteers, who were treated according to the
Helsinki protocol for biomedical experimentation, gave their informed
consent to the use of blood samples for research purposes. Platelet rich
plasma (PRP) was prepared by centrifugation of citrated blood at 210g
for 20 min. Aliquots (500 μL) of PRP were added into aggregometer
(Chrono-log 4902D) cuvettes, and aggregation was recorded as in-
creased light transmission under continuous stirring (1000 rpm) at
37 °C for 10 min after addition of the stimulus. Collagen at submaximal
concentration (0.8À1.5 μg/mL) was used as a platelet activator in PRP.
Compounds under study were preincubated with PRP 10 min before
addition of the stimulus (collagen). Vehicle alone (0.5% DMSO) added
to PRP did not affect platelet function in control samples. At least five
experiments for each compound were performed.
’ ASSOCIATED CONTENT
S
Supporting Information. Elemental analysis data for the
b
target compounds. This material is available free of charge via the
Internet at http://pubs.acs.org.
’ AUTHOR INFORMATION
Corresponding Author
*Phone: +39 011 6707670. Fax: +39 011 6707286. E-mail:
alberto.gasco@unito.it.
’ ABBREVIATIONS USED
NSAIDs, nonsteroidal anti-inflammatory drugs; PRP, platelet
rich plasma; NO, nitric oxide; sGC, soluble guanylate cyclase;
COX, cyclooxigenase; ODQ, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-
1-one; ASA, acetylsalicylic acid; THP, tetrahydropyranyl; DIAD, di-
isopropyl azodicarboxylate; PPTS, pyridinium p-toluensulfonate
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